INACTIVATION OF STREPTOCOCCUS-PYOGENES EXTRACELLULAR CYSTEINE PROTEASE SIGNIFICANTLY DECREASES MOUSE LETHALITY OF SEROTYPE M3 AND M49 STRAINS

Cysteine proteases have been implicated as important virulence factors in a wide range of prokaryotic and eukaryotic pathogens, but little d irect evidence has been presented to support this notion. Virtually al l strains of the human bacterial pathogen Streptococcus pyogenes expre ss a highly conserved extracellular cysteine protease known as strepto coccal pyrogenic exotoxin B (SpeB). Two sets of isogenic strains defic ient in SpeB cysteine protease activity were constructed by integratio nal mutagenesis using nonreplicating recombinant plasmids containing a truncated segment of the speB gene. Immunoblot analyses and enzyme as says confirmed that the mutant derivatives were deficient in expressio n of enzymatically active SpeB cysteine protease. To test the hypothes is that the cysteine protease participates in host mortality, we asses sed the ability of serotype M3 and M49 wild-type strains and isogenic protease-negative mutants to cause death in outbred mice after intrape ritoneal inoculation. Compared to wild-type parental organisms, the se rotype M3 speB mutant lost virtually all ability to cause mouse death (P < 0.00001), and similarly, the virulence of the M49 mutant was detr imentally altered (P < 0.005). The data unambiguously demonstrate that the streptococcal enzyme is a virulence factor, and thereby provide a dditional evidence that microbial cysteine proteases are critical in h ost-pathogen interactions.